The goal of the basic research proposed here is to understand the molecular mechanisms underlying critical adaptive changes occurring in natural populations of fish after long-term, multi-generational exposure to mixtures of contaminants at Superfund sites. The research applies innovative molecular approaches in an ecological context to understand chemical-induced evolutionary changes in signaling pathways implicated in the response to Superfund chemicals. The studies focus on the Atlantic killifish Fundulus heteroclitus, a unique model system for integrated investigation of ecological and mechanistic questions concerning the impact of chemicals at Superfund sites. At several locations along the Atlantic coast, including the New Bedford Harbor (NBH), MA Superfund site, populations of killifish have evolved resistance to the highly toxic, dioxin-like (non-ortho-substituted) polychlorinated biphenyls (PCBs) and other chemicals that act through the aryl hydrocarbon receptor (AHR). Our recent studies show that NBH killifish also appear to have reduced sensitivity to non-dioxin-like (ortho-substituted) PCBs?the most abundant components of PCB mixtures?but the mechanisms involved are not known. While allelic variation at killifish AHR loci has been associated with resistance to dioxin-like PCBs, recent studies have implicated another AHR pathway member, AHR- interacting protein (AIP; also known as Ara9 or XAP2) as the strongest candidate for a resistance gene in killifish populations exhibiting reduced sensitivity to dioxin-like compounds. The central hypothesis is that the evolution of resistance to dioxin-like PCBs involves additive or epistatic interactions between multiple components of the AHR pathway, including AIP and one or more of the four killifish AHRs. To test this hypothesis, in Aim 1 we propose to use CRISPR-Cas9 genome-editing technology to generate null and hypomorphic alleles of AIP in killifish and zebrafish and to use them to test hypotheses about the role of AIP in the mechanism of resistance to dioxin-like compounds. In Aim 2, we will identify AIP single nucleotide polymorphisms (SNPs) associated with the resistant phenotype in PCB-resistant killifish, and use CRISPR- Cas9-mediated, single-nucleotide genome editing of zebrafish AIP to study the role of these AIP SNPs in the mechanism of PCB-resistance. Aim 3 will elucidate the mechanism of resistance to ortho-PCBs and, in particular, whether it is a) secondary to defects in AIP or AHR or b) an independently-evolved phenotype. We will also investigate possible mechanisms of o-PCB toxicity involving altered signaling through nuclear receptors such as PXR and PPAR that interact with AIP or exhibit cross-talk with AHR. The proposed research represents a unique opportunity to establish a novel mechanism of population-level effects of pollutants and will advance our understanding of the long-term impact of chemical mixtures on ecological systems.